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http://pen.sagepub.com/content/early/2013/06/10/0148607113491608The online version of this article can be found at:

DOI: 10.1177/0148607113491608

published online 11 June 2013JPEN J Parenter Enteral NutrYongjia Feng, Pele Browner and Daniel H. Teitelbaum

Parenteral NutritionEffects on Varying Intravenous Lipid Emulsions on the Small Bowel Epithelium in a Mouse Model of

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Journal of Parenteral and Enteral

Nutrition

Volume XX Number X

Month 2013 1 –12

© 2013 American Society

for Parenteral and Enteral Nutrition

DOI: 10.1177/0148607113491608

jpen.sagepub.com

hosted at

online.sagepub.com

2013 Premier Research Paper

Intravenous (IV) lipids should comprise between 30% and

50% of all nonnitrogen calories. However, the refinement and

development of IV fat emulsions (IVFEs) have been complex.1

All IVFEs provided in the United States are derived from soy-

bean oil and are predominately long-chain triglyceride (LCT)

ω-6 polyunsaturated fatty acids (FAs) and thus, in certain set-

tings, have been shown to drive a proinflammatory response.

To address these potential shortcomings, other sources of

fat emulsions (FEs), including medium-chain triglycerides

(MCTs), can reduce the total amount of LCTs delivered to the

patient. Additional advantages of MCTs include greater solubi-

lization of the lipid component, decreased accumulation of lip-

ids in the liver, and improved clearance and reduced production

of proinflammatory mediators.2,3 In addition, use of MCTs

may actually improve immune function by decreasing the lev-

els of tumor necrosis factor–α (TNF-α).4 Another strategy is

the replacement of soybean-predominant FE with an olive oil–

predominant fat. Use of agents with 80% olive oil can reduce a

patient’s exposure to ω-6 FAs. Furthermore, olive oils contain

a blend of ω-3, ω-6, and ω-9 FAs and other antioxidants,

including polyphenols. Another alternative FE strategy has

been to use fish oil. A fish oil FE is composed of almost entirely

ω-3 FAs and may also prevent a proinflammatory state.5

The ideal IVFE remains elusive, and gaining the ideal bal-

ance of FAs may have tremendous implications for patient out-

comes, including mortality.6,7 Comparisons of FAs have

identified that even clinically approved IVFEs can lead to

491608 PENXXX10.1177/0148607113491608<italic>Journal of Parenteral and Enteral Nutrition /</italic> Vol. XX, No. X, Month XXXXFeng et alresearch-article2013

From the Section of Pediatric Surgery, Department of Surgery, the

University of Michigan Medical School and the C. S. Mott Children’s

Hospital, Ann Arbor, Michigan.

Financial disclosures: The study was supported by a grant from the

Baxter Healthcare Corp. Baxter did not have an influence on the contents

of this article or preview its contents.

Supplementary material for this article is available on the Journal of

Parenteral and Enteral Nutrition website at http://pen.sagepub.com/

supplemental.

Received for publication January 2, 2013; accepted for publication May

6, 2013.

Corresponding Author:

Yongjia Feng, MD, PhD, Section of Pediatric Surgery, University of

Michigan, Mott Children’s Hospital, 1540 E. Hospital Dr, SPC 4211,

Ann Arbor, MI 48109-4211, USA.

Email: yongfeng@umich.edu.

Effects on Varying Intravenous Lipid Emulsions on the

Small Bowel Epithelium in a Mouse Model of Parenteral

Nutrition

Yongjia Feng, MD, PhD; Pele Browner, BS; and Daniel H. Teitelbaum, MD

AbstractBackground: Injectable fat emulsions (FEs) are a clinically dependable source of essential fatty acids (FA). ω-6 FA is associated with

an inflammatory response. Medium-chain triglycerides (MCT, ω-3 FA), fish oil, and olive oil are reported to decrease the inflammatory

response. However, the effect of these lipids on the gastrointestinal tract has not been well studied. To address this, we used a mouse

model of parenteral nutrition (PN) and hypothesized that a decrease in intestinal inflammation would be seen when either fish oil and

MCT or olive oil were added. Methods: Three FEs were studied in adult C57BL/6 mice via intravenous cannulation: standard soybean-

based FE (SBFE), 80% olive oil -supplemented FE (OOFE), or a combination of a soybean oil, MCT, olive oil, and fish oil emulsion

(SMOF). PN was given for 7 days, small bowel mucosa-derived cytokines, animal survival rate, epithelial cell (EC) proliferation and

apoptosis rates, intestinal barrier function and mucosal FA composition were analyzed. Results: Compared to the SBFE and SMOF

groups, the best survival, highest EC proliferation and lowest EC apoptosis rates were observed in the OOFE group; and associated with

the lowest levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β expression. Jejunal FA content showed higher levels of

eicosapentaenoic and docosapentaenoic acid in the SMOF group and the highest arachidonic acid in the OOFE group. Conclusion: The

study showed that PN containing OOFE had beneficial effects to small bowel health and animal survival. Further investigation may help

to enhance bowel integrity in patients restricted to PN. (JPEN J Parenter Enteral Nutr. XXXX;xx:xx-xx)

Keywordsintravenous lipids; parenteral nutrition; epithelial cell apoptosis; epithelial cell proliferation; ω-3 lipids; ω-6 lipids; medium-chain

triglycerides; fish oil

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2 Journal of Parenteral and Enteral Nutrition XX(X)

essential FA deficiencies, and thus further optimization of for-

mulas is needed.8 Use of such alternative FEs has shown prom-

ising effects in reducing a proinflammatory injury in the liver.9

However, although many of these now commercially available

FEs have been studied in terms of their system effects, little is

known regarding the action of these fats on the intestinal epi-

thelium. This has significant implications as the administration

of parenteral nutrition (PN) results in significant loss of intes-

tinal epithelial barrier function (EBF) and profound intestinal

mucosal atrophy10; these changes have been associated with a

marked increase in the mucosal expression of several proin-

flammatory cytokines.11 However, it is unknown if different

IVFEs can influence these adverse changes. To address this,

we used a mouse PN model and hypothesized that a decrease

in intestinal inflammatory-linked cytokines and intestinal atro-

phy would be seen when alternative IVFEs were added to

existing soybean lipid emulsions, thereby reducing the total

exposure to soybean oil.

Methods

PN Animal Model

Male specific pathogen-free (SPF) C57BL/6J mice (10–12

weeks old; Jackson Laboratory, Bar Harbor, ME) were main-

tained under temperature-, humidity-, and light-controlled con-

ditions. Mice were initially fed ad libitum with standard mouse

chow and water and allowed to acclimate for 1 week prior to

surgery. During the administration of IV solutions, mice were

housed in metabolic cages to prevent coprophagia. Studies

conformed to guidelines for care established by the University

Committee on Use and Care of Animals at the University of

Michigan, and protocols were approved by that committee

(No. 03986). Methods of cannulation and PN infusion were

similar to that previously described.12-14 The control group

received an IV crystalloid solution (dextrose 5% in 0.45 NS

with 20 mEq KCl/L) at 0.3 mL/h and standard laboratory

mouse chow. The PN groups received an IV PN solution at 7.2

mL/24 hours but were allowed water ad libitum. All study

groups had complete nutrient supplementation, with all essen-

tial vitamins, trace elements, and minerals being supplied;

details of caloric delivery have been published in this journal.15

All animals received similar daily energy and nitrogen delivery

and were euthanized at 7 days using CO2. The small intestine

was exposed from the ligament of Treitz. We discarded the first

5 cm of jejunum and then took 2 cm of jejunum for FA analy-

sis, 1 cm of jejunum for RNA extraction, another 2 cm for his-

tology, and 4 cm for Ussing chamber testing (Suppl. Fig. S1).

IVFE and Study Masking

Three IVFEs were studied (n = 8/group) in adult C57BL/6

mice via IV cannulation: standard soybean-based FE (SBFE),

olive oil–supplemented FE (OOFE; composed of 80% olive oil

and 20% soybean oil), and a combination soybean oil (30%),

medium-chain triglycerides (30%), olive oil (25%), and fish oil

emulsion (15%) (SMOF). To ensure a nonbiased approach, the

FEs were initially masked so that investigators did not know

(ie, were blinded) the identity of the FEs until the data analysis

was completed. These 3 groups were compared with a sham or

chow-fed group of mice. All FEs were derived from commer-

cial products as follows: SBFE (Intralipid; Fresenius Kabi,

Uppsala, Sweden), OOFE (Clinoleic; Baxter Healthcare,

Deerfield, IL), and SMOF (SMOFlipid, Fresenius Kabi). Each

FE was studied at 3 concentrations, comprising 10%, 20%, or

30% of total daily energy delivery as fat. Compensation of

energy delivery between groups was provided by the addition

or subtraction of dextrose to ensure each group was isocaloric,

isonitrogenous, and isovolemic. All volumes, energy delivery,

and nitrogen concentrations were balanced between all study

groups. Furthermore, all solutions were compounded by a ster-

ile, class 100,000 facility at our university (HomeMed Infusion

Company) as a 3:1 solution.

Intestinal Morphology Assessment

Villus height and crypt depth were measured in at least 20

well-oriented full-length crypt-villus units per specimen and

averaged. Data were analyzed using commercially available

digital image analysis software NIS-Elements, AR 3.0 (Nikon

Instruments Inc, Melville, NY).

Epithelial Cell Proliferation and Apoptosis

For epithelial cell (EC) proliferation, immunofluorescent stain-

ing for proliferating cell nuclear antigen (PCNA) was per-

formed as previously described.16 Results are expressed as the

ratio of the number of PCNA-positive cells to the total number

of crypt cells. A total of 15–20 crypts were measured for each

mouse. EC proliferation was also measured with BrdU stain-

ing, as previously described.17 Immunofluorescent staining for

active caspase-3 was performed to assess EC apoptosis, as pre-

viously described.10 Results are expressed as an apoptotic

index (% active caspase-3–positive cell number/villi cell num-

ber, using a mean of 10–15 villi/mouse).

Real-Time Polymerase Chain Reaction

RNA extraction of jejunum mucosal scrapings was processed

as previously described.10 Oligomers were designed using an

optimization program (www.premierBiosoft.com). Real-time

polymerase chain reaction was performed and normalized to

β-actin.18

Immunofluorescence Microscopy

A 0.5-cm section of fresh jejunum (~10 cm from the ligament

of Treitz) was harvested and immunofluorescence staining was

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Feng et al 3

performed as previously described.19 Fluorescence was ana-

lyzed by using an Olympus BX-51 upright light and fluores-

cence microscope (Olympus, Center Valley, PA), and images

were stacked using Nikon image software (Nikon, Tokyo,

Japan) and processed using Adobe Photoshop CS4 (Adobe,

San Jose, CA).

Epithelial Barrier Function

Full-thickness jejunal tissue was mounted in Ussing chambers.

Assessment of EBF was measured using previously described

methods.11 Jejunum transepithelial resistance (TER) was deter-

mined by measurements of spontaneous voltage and isoelectric

current using the Ohms law after a 30-minute equilibration

period and was expressed Ω·cm2.

FA Analysis

After mice jejunum tissue, which had been frozen at −80°C,

was slowly warmed, a 150-g piece was cut and transferred to a

blood collection tube (100 × 16 mm). The weight (mg) of the

tissue piece was recorded and then put on ice. Then, 1.0 mL of

cooled phosphate-buffered saline was added to the tube, and

the sample was homogenized with an Omni homogenizer and

Omni-Tips (Omni international homogenizer company,

Kennesaw, GA) for 30 minutes and then put back on ice. Next,

100 µL of jejunum homogenate was transferred to a disposable

glass tube (16 × 100 mm; Pyrex with Teflon-lined screw caps).

To these tubes, 20 µL of tricosanoic acid (C23:0, 0.989 mg/

mL) as an internal standard and 2000 µL of methanol-benzene

4:1 (v/v) were then added. The tubes were vortexed gently.

Tubes were placed in a dry ice bath for 10 minutes. Then, 250

µL of acetyl chloride was added to each tube while on dry ice,

the caps were tightly closed, and the tube rack was transferred

at room temperature (RT) until the reaction mixture had melted.

The tubes were then vortexed gently. The tubes were purged

with nitrogen gas, with the Teflon-lined caps tightly closed

again, and subjected to RT for 24 hours. The tubes were cooled

in an ice bucket, and then 5 mL of 6% K2CO

3 solution was

added slowly to stop the reaction and neutralize the mixture.

The reaction mixture was vortexed and then centrifuged at

3000 rmp for 20 minutes (25°C) to separate layers. An aliquot

(150–200 µL) was removed by gently tilting the tubes and

drawing off the benzene layer with a hand pipettor, using the

long-tailed pipet tips. The contents were transferred in the sam-

ple vials (amber color with glass inserts) for gas chromatogra-

phy (GC) analysis. For each sample, peaks were identified

based on the retention time compared with the corresponding

reference standards; peak area was determined for each FA

methyl ester, including C23:0 methyl ester (as internal stan-

dard), from the GC chromatograph.20 FA methyl ester concen-

trations in the sample were normalized and calculated relative

to that of the internal standard (C23:0) and tissue amount.

Data Analysis

Data were expressed as mean ± standard deviation (SD).

Statistical analysis employed paired t tests for comparison of 2

means and a 1-way analysis of variance (ANOVA) for com-

parison of multiple groups (with a Bonferroni post hoc analysis

to assess statistical differences between groups), and 2-way

ANOVA test was used for categorical data (Prism software;

GraphPad Software, San Diego, CA). Statistical significance

was defined as P < .05.

Results

Preliminary studies were performed with FE concentrations

being studied at 10%, 20%, and 30%. Due to the complexity of

reporting the results, we believed that the most clinically rele-

vant percentage that would report the bulk of the results was

30% of total daily calories as fat, as this approximates a typical

level added to PN solutions. As well, this percentage was most

similar to that previously studied by our research group.

Additional results at the 10% and 20% FE concentrations are

reported in Table 1.

Survival and Body Weight

Survival rates were compared with mice in each group (Figure

1A). Sham mice had a 100% survival after the 7-day study

period. Distinct differences in survival were noted between the

FE groups. Mice in the SBFE group had an approximately 65%

survival, which was similar to our previously reported rate of

70%.16,21,22 Mice in the SMOF group, although not statistically

different from the SBFE group, had a lower survival rate

(55%). Interestingly, mice in the OOFE group had the highest

survival rate (100%), and rates were identical to the 100% sur-

vival of the chow-fed controls.

Body weight loss occurred in all PN mice, as has been

reported previously (Figure 1B).10,21 The range of weight loss

was 4–5 g over the 1-week infusion period, and although sig-

nificantly greater than sham (chow-fed) mice, no differences

were identified between the FE groups.

Intestinal Morphology and Epithelial Cell

Turnover

As previously reported, administration of PN led to a signifi-

cant degree of intestinal atrophy. Table 1 shows that villus

height decreased significantly in all PN-treated groups com-

pared with the sham group. The greatest decline was found in

the SBFE group. Although lower than the sham group, the

SMOF and OOFE groups both had longer villus lengths com-

pared with the SBFE group. No significant differences were

seen in the crypt depth between the study groups.

Since PN-associated villus atrophy is due to a loss of EC

proliferation and an increase in EC apoptosis,23,24 we

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4 Journal of Parenteral and Enteral Nutrition XX(X)

investigated both. Figure 2 shows EC apoptosis rates, as

measured by active caspase-3 staining in the 30% FE groups

compared with enteral controls (sham). Note that EC apopto-

sis rates rose significantly (3-fold greater than the sham

group) in the SBFE group. Rates of apoptosis were

somewhat lower in the SMOF group (not significantly dif-

ferent), and levels declined significantly in the OOFE group

to rates similar to those seen in the sham group.

EC proliferation rates were examined using 2 methods,

PCNA (Figure 3A) and BrdU (Figure 3B) immunofluores-

cent staining. Both methods showed similar findings, includ-

ing a significant decline in EC proliferation rates between all

study groups vs shams. However, between the 3 FE groups,

differences were seen. Of note, EC proliferation rates were

significantly higher in the 30% OOFE group compared with

the SBFE group (for PCNA), and when evaluated by BrdU

staining, the EC proliferation rates in OOFE mice were sig-

nificantly higher compared with both the SBFE and SMOF

groups.

To better address the mechanisms that may have led to these

changes, several gene factors associated with EC proliferation

and apoptosis were examined (Figure 4). Interestingly, despite

the SBFE group having the lowest EC proliferation rate and

greatest degree of atrophy, messenger RNA (mRNA) expres-

sion of EGF, although not significantly different, was actually

highest in the SBFE group and lowest in the OOFE group. The

EGF receptor, ErbB1, significantly decreased in the SMOF and

OOFE groups, and epiregulin and transforming growth factor–

α (TGF-α) significantly declined in the OOFE group (Figure

4C,D). Thus, the precise mechanism that led to improved EC

proliferation is not clearly identified.

Intestinal Muscosal Gene Expression

mRNA expression of several proinflammatory factors and

junctional proteins was examined in each study group, and dis-

tinct differences were noted (Figures 5 and 6). SBFE adminis-

tration was associated with the upregulation of TNF-α, TNF

receptor 1 (TNFR1), interleukin (IL)–1β, and IL-6. In addition,

SBFE was associated with an upregulation of Toll-like recep-

tor 4 (Tlr4). Somewhat surprisingly, although the SMOF group

was associated with slightly lower proinflammatory cytokine

abundances, SMOF was associated with an increase in TNF-α

abundance. In contrast, the OOFE group was associated with a

Table 1. Summary of Key Clinical and Cytokine Data.

Group Sham SBFE 10% SMOF 10% OOFE 10% SBFE 20% SMOF 20% OOFE 20% SBFE 30% SMOF 30% OOFE 30%

Crypt depth 22.2 ± 1.9 23.5 ± 1.1 24.1 ± 0.6 23.5 ± 1.1 22.8 ± 0.5 17.2 ± 1.7 28.6 ± 1.5 24.9 ± 1.3 16.3 ± 0.8 25.1 ± 2.1

Villus height 251.3 ± 74 160.9 ± 18.0 218.8 ± 32.4 196.3 ± 22.9 206.1 ± 51.5 192.2 ± 5.5 189.0 ± 14.9 165.7 ± 16.7 168.5 ± 23.1 183.0 ± 14.7

PCNA 0.42 ± 0.03 0.14 ± 0.03 0.12 ± 0.03 0.24 ± 0.04 0.24 ± 0.02 0.25 ± 0.05 0.25 ± 0.05 0.22 ± 0.04 0.24 ± 0.05 0.28 ± 0.02

TNF-αa 0.30 ± 0.07 0.49 ± 0.30 0.95 ± 0.79 0.44 ± 0.21 0.42 ± 0.22 0.38 ± 0.12 0.19 ± 0.08 0.42 ± 0.15 0.69 ± 0.16 0.34 ± 0.20

TNF-R1a 67.0 ± 12.8 96.4 ± 18.8 116.6 ± 36.5 73.0 ± 22.1 111.9 ± 29.6 74.8 ± 11.6 52.3 ± 21.5 134.7 ± 39.6 79.5 ± 18.7 43.6 ± 6.2

IL-1βa 3.37 ± 2.22 4.84 ± 3.89 3.89 ± 2.60 2.46 ± 1.44 6.08 ± 4.28 2.39 ± 2.48 1.30 ± 1.03 5.87 ± 3.04 2.66 ± 1.49 1.67 ± 1.37

IL-6a 0.25 ± 0.12 0.30 ± 0.23 0.73 ± 0.25 0.16 ± 0.16 0.26 ± 0.15 0.22 ± 0.09 0.15 ± 0.07 0.45 ± 0.23 0.11 ± 0.11 0.09 ± 0.04

INF-γa 0.19 ± 0.09 0.51 ± 0.03 1.33 ± 0.93 0.43 ± 0.35 0.14 ± 0.05 0.60 ± 0.29 0.18 ± 0.05 0.26 ± 0.19 0.26 ± 0.11 0.22 ± 0.15

Tlr4a 0.59 ± 0.09 1.01 ± 0.30 1.41 ± 0.90 0.43 ± 0.20 0.56 ± 0.28 0.19 ± 0.09 0.50 ± 0.33 1.08 ± 0.36 0.68 ± 0.23 0.57 ± 0.32

IL, interleukin; INF-γ, interferon γ; OOFE, 80% olive oil–supplemented fat emulsion; PCNA, proliferating cell nuclear antigen; SBFE, standard soy-

bean-based fat emulsion; SMOF, combination of soybean oil, medium-chain triglycerides, olive oil, and fish oil emulsion; TNF, tumor necrosis factor.aMultiplied by 1000.

Figure 1. Different fat emulsions significantly affected mouse

survival. (A) Date of death was recorded for mice over the 7 days

of study. The results are recorded on a survival curve. Of note

was a significantly improved survival rate in the 30% OOFE

group (100% survival) compared with the SBEF and SMOF

groups. (B) Body weight was recorded before and after the 1

week of PN administration. No significant differences were noted

between the PN groups. OOFE, 80% olive oil–supplemented

fat emulsion; PN, parenteral nutrition; SBFE, standard soybean-

based fat emulsion; SMOF, combination of soybean oil, medium-

chain triglycerides, olive oil, and fish oil emulsion.

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Feng et al 5

significant decline in all proinflammatory cytokines and Tlr4,

which were elevated in the SBFE group. No significant changes

were noted in the abundance of interferon (INF)–γ in any of the

treatment groups.

mRNA expression of various tight junction markers was

measured (Figure 6A–C). Levels of each tight junction factor

varied with the type of FE. Abundance of occludin and ZO-1

levels increased in the SBFE group, with a significant decline

in the SMOF and OOFE groups. Similarly, claudin-2 was also

increased in the SBFE group (approximately 3-fold higher than

the sham group; Figure 6C), as previously reported,21,25 and

2-fold higher than sham mice in the SMOF and OOFE groups.

Claudin-15, however, was associated with a significant decline

in all FE groups (Figure 6C). The overall increase in claudin-2

is consistent with its overall pore-forming action with a resul-

tant loss of barrier function. As well, not shown was a global

decrease in claudin-7 abundance in all FE groups.

EBF and Intestinal FA Content

Barrier function was then measured in Ussing chambers

(Figure 6D). Interestingly, despite the increase in occludin and

ZO-1, TER results demonstrated a significant decline in levels

for all FE groups, and there were no differences noted between

the groups.

Finally, tissue was collected to analyze the FA content in

proximal intestinal tissue. Table 2 shows the summary of these

results at the 30% FE level, with total levels essentially the

same between the groups. In general, trends followed regard-

less of the dosage of FE given. The results showed that the

sham (enterally fed) group had somewhat higher levels of

short-chain FAs (SCFAs) than the SBFE group but similar

oleic acid levels. The sham mice had the highest levels of lin-

oleic and linolenic acids and lowest levels of arachidonic acid

(AA). Higher levels of eicosapentaenoic acid (EPA) and doc-

osapentaenoic acid (DPA) levels were found in the sham and

SMOF groups compared with the SBFF and OOFE groups.

Interestingly, the OOFE group exhibited the highest percent-

age of AA, irrespective of the dosage level.

Discussion

In this article, we examined 3 commercially available IVFEs

regarding their effect on intestinal mucosal gene expression,

morphology, epithelial cell turnover, and overall systemic

effect on body weight and survival. Interestingly, we found dis-

tinct and significant changes between these 3 compounds.

Overall weight of the mice was not different between groups,

and outcomes at each percentage of FE varied somewhat

between groups. However, when focusing the results on the

30% of energy via the FE group, survival patterns were dra-

matically different. In particular, use of OOFE resulted in a

nearly 100% survival, whereas SMOF and SBFE resulted in

far lower survival rates. Although surprising, other groups

have also identified issues with the use of soybean-based lip-

ids. A comparison of survival between fish oil vs soybean FE

in a septic rat model demonstrated a significant improvement

in survival in the fish oil group.26

An area that has not been studied is the effect that FE prod-

ucts might have on intestinal physiology. Although one might

suspect that matched energy delivery would suffice to yield

Figure 2. EC apoptosis rates were markedly increased in the

SBFE PN group. Apoptosis was measured with active caspase-3

staining in small bowel epithelium. (A) Representative active

caspase-3 immunofluorescence images from each study group.

(B) Apoptosis index is the ratio of active caspase-3 cells to the

total number of epithelial cells. Note that the SBFE group had

the highest EC apoptosis index and OOFE mice had the lowest

apoptosis index compared with the SBFE and SMOF groups,

with levels reaching those of the sham group. EC, epithelial cell;

OOFE, 80% olive oil–supplemented fat emulsion; PN, parenteral

nutrition; SBFE, standard soybean-based fat emulsion; SMOF,

combination of soybean oil, medium-chain triglycerides, olive

oil, and fish oil emulsion.

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6 Journal of Parenteral and Enteral Nutrition XX(X)

similar outcomes in each group, striking differences were

noted. When examined at the 30% FE dosing, EC prolifera-

tion was significantly higher and EC apoptosis rates were sig-

nificantly lower in the OOFE group, with a resultant partial

prevention of PN-associated mucosal atrophy. mRNA analysis

demonstrated several changes in gene expression that appeared

somewhat contrary to the EC findings. This included an upreg-

ulation of TCF4/LEF transcription factors cyclin D1 and

c-Myc in the SBFE group, particularly at the 30% concentra-

tion. In addition, a decline in the stem cell marker LGR5 and

declines in cyclin D1 and c-Myc were seen in the OOFE group.

It is possible that the upregulation in the SBFE group is a com-

pensatory attempt to prevent the adverse effects (atrophy)

occurring with this FE. It may also be an attempt to counter the

proinflammatory state associated with the infusion of SBFE.

Our findings showed a trend toward a proinflammatory

cytokine state within the intestinal mucosa in mice given

SBFE, with reduced levels of these cytokines in the SMOF

group and even lower levels in OOFE-treated mice. This

included a rise in IL-1β and IL-6. We were surprised to see the

increase in TNF-α abundance in the SMOF group. Although

this lipid is thought to prevent a proinflammatory state, the eti-

ology of this elevation is unknown, and it is possible that the

unique immune system of the gastrointestinal (GI) tract may

respond differently to this combination of lipids or may be due

to the type of substrate provided to either immunocyte, the

upregulation of Tlr4, or the altered intraluminal bacteria.22

Although not examined within the GI tract, several studies

have pointed to a proinflammatory state with soybean oil. In a

study examining fish oil vs soybean oil FE, a significant

Figure 3. EC proliferation rates in crypt were markedly decreased with PN administration; EC proliferation rates were measured

with PCNA and BrdU staining and expressed by the ratio of positive cells numbers compared with the total crypt ECs numbers. (A)

Representative images of PCNA staining. (B) Ratio of PCNA-positive cells to total number of crypt cells. Note that all PN groups showed

a reduced PCNA expression compared with the sham group, and improved EC PCNA staining was observed in the OOFE group vs SBFE

and SMOF groups. BrdU staining (C, D) showed similar results. EC, epithelial cell; OOFE, 80% olive oil–supplemented fat emulsion;

PCNA, proliferating cell nuclear antigen; PN, parenteral nutrition; SBFE, standard soybean-based fat emulsion; SMOF, combination of

soybean oil, medium-chain triglycerides, olive oil, and fish oil emulsion.

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Feng et al 7

improvement in lymphocyte function was identified.26 In

another study between fish oil vs soybean oil, fish oil led to an

increase in TH2-based cytokine formation within spleno-

cytes.27 In a clinical study of preterm infants, a reduction of

IL-6 was observed in patients fed olive oil vs soybean FE.28

Finally, human leukocytes showed a marked improvement in

lymphocyte activation in olive oil FE exposed vs soybean FE

cells.29 Together, this suggests improved immune function and

a decline in proinflammatory cytokines with either olive oil–

or fish oil–based FEs. The precise mechanism by which the

OOFE group had markedly improved survival is unknown. It is

quite possible that this could be due to an effect of the unique

antioxidant effect or differences in polyphenol composition. It

is also possible that the reduced levels of proinflammatory

cytokines in this lipid group may be a major contributory

mechanism.

Barrier function has been shown to significantly decline

with administration of PN.11,12 The assessment of tight junction

factors, occludin, ZO-1, claudin-2, and claudin-15, demon-

strated some differences between the groups, with a moderate

increase in occludin, claudin-2, and ZO-1 in the SBFE group

and a decline in all FE groups for claudin-15. Interestingly,

TER was noted to be significantly decreased in all FE groups,

regardless of the mRNA changes in tight junction factors. This

Figure 4. Changes in ErbB ligands and EGF receptor (EGFR) expression with PN administration. Messenger RNA expression of ErbB

ligands and the EGFR was determined with real-time polymerase chain reaction, and expressed as a ratio to β-actin. (A) EGF expression:

note the slight decline in the SMOF and OOFE groups; however, no changes were significant. (B) EGF expression: note the significant

decline in the SMOF and OOFE groups. (C) Epiregulin expression: note the significant decline in the OOFE group. (D) TGF-α expression:

note the significant decline in the OOFE group. EGF, epidermal growth factor; OOFE, 80% olive oil–supplemented fat emulsion; PN,

parenteral nutrition; SBFE, standard soybean-based fat emulsion; SMOF, combination of soybean oil, medium-chain triglycerides, olive

oil, and fish oil emulsion; TGF-α, transforming growth factor–α.

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8 Journal of Parenteral and Enteral Nutrition XX(X)

Figure 5. Proinflammatory cytokines and signaling receptors were increased with PN but downregulated with OOFE. Proinflammatory

cytokines and the selected TNF-α receptor R1 (TNFR1) were measured with real-time polymerase chain reaction and corrected for

expression by measuring β-actin messenger RNA. (A) TNF-α expression showed that the SMOF group had the highest levels of TNF-α

(P < .01) compared with either the SBFE or OOFE group. TNFR1 expression was highest in the SBFE group (P < .01), with slightly

reduced levels in the SMOF group and significantly lower levels in the OOFE group (P < .01). (B) IL-6 and IL-1β expression were both

significantly increased in the SBFE group. Levels of these cytokines were reduced in the SMOF group to that of a similar range to sham

mice, and levels were lowest in the OOFE group and declined below that of sham mice (P < .01). (C) INF-γ levels did not significantly

change between study groups, whereas Tlr4 expression significantly increased in the SBFE group compared with sham mice and was

significantly decreased in the OOFE group (P < .01). IL, interleukin; INF-γ, interferon γ; OOFE, 80% olive oil–supplemented fat

emulsion; PN, parenteral nutrition; SBFE, standard soybean-based fat emulsion; SMOF, combination of soybean oil, medium-chain

triglycerides, olive oil, and fish oil emulsion; TGF-α, transforming growth factor–α; Tlr4, Toll-like receptor 4.

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Feng et al 9

Figure 6. Jejunal barrier function was decreased with PN administration. EBF was measured by the abundance of selected tight

junction factors at the mRNA level. EBF was also measured detecting TER in full-thickness jejunal tissue. (A) mRNA levels of tight

junction factors ZO-1 and occludin rose modestly in the SBFE group and significantly declined in the SMOF and OOFE groups

(P < .05). (B) Claudin-2 mRNA abundance rose significantly in all PN groups, with the highest abundance noted in the SBFE group

(P < .05). Claudin-15 mRNA levels were significantly lower than the sham group in all FE study groups (P < .05). (C) TER measures

showed a significant decline in all FE study groups compared with the sham group (P < .05). EBF, epithelial barrier function; FE,

fat emulsion; mRNA, messenger RNA; OOFE, 80% olive oil–supplemented fat emulsion; PN, parenteral nutrition; SBFE, standard

soybean-based fat emulsion; SMOF, combination of soybean oil, medium-chain triglycerides, olive oil, and fish oil emulsion; TER,

transepithelial resistance.

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10 Journal of Parenteral and Enteral Nutrition XX(X)

is most likely due to the fact that EBF correlates better with the

actual structural integrity of these factors,30 and these could all

be disrupted with the measured increase in proinflammatory

markers and/or loss of mucosal growth factors in the PN

groups.

Mechanisms that might account for these differences are

not known. One of the more intriguing findings was the marked

decline in proinflammatory cytokines in the OOFE group, yet

a marked decline in the abundance of several growth factors. It

has been increasingly appreciated over the past 5 years that

there is a tight transactivation between TNF-α and EGF.31

However, with PN administration, our group has previously

reported an increase in TNF-α and a marked loss of EGF and

several EGF family members.22 It is possible that the increased

mRNA abundance of TNF-α in the SBFE and SMOF groups

and loss of EGF in the OOFE group led to the uniform loss of

EBF in all FE study groups.

The changes in mucosal FAs measured in the present study

were interesting and may have significant physiologic corre-

lates. For instance, the marked increase in AA in the OOFE

group could result in enhanced protection of the host, despite

the loss of EBF. Recently, enhancement of AA levels within the

small bowel was shown to improve barrier function in an acute

ischemia model in suckling piglets.32 In contrast, the increase

in EPA in the SMOF group might help protect the intestine

from the increase in proinflammatory cytokines, as EPA has

also been shown to enhance EBF.32

However, the combination of lipids and the precise mecha-

nisms that have resulted in the present findings are complex

and not fully determined in this study. It is also not clear how

differing FAs may have affected the intestinal barrier function

in our study. It has been reported that polyunsaturated FAs

induce intracellular acidosis, which can decrease the intracel-

lular adenosine triphosphate level and inhibit Ca+2-ATPase.

The increase in the calcium level activates actomyosin con-

traction through the activation of cytoskeletal stabilization or

through other processes leading to the opening of the tight

junctions.33 Some investigators have shown that DHA and

EPA have a positive effect on impaired EBF induced by IFN-γ

and TNF.34 However, this was not shown in the current study.

Therefore, this suggests that other factors may contribute to

changes in both EC proliferation and injury of barrier function

in this PN model, and future studies will need to be done.

One important issue was that we selected a 30% lipid dose

to make this most relevant to the clinical delivery of nutrition.

This amount of lipid is higher than what most mouse diets

Table 2. Fatty Acid Content From Jejunal Mucosal Tissue From Mice.

Fatty Acid Sham SBFE 30% SMOF 30% OOFE 30%

Total content of fatty acids from jejunal

mucosa, mg/g of mucosal tissue

13.97 ± 4.07 (n = 6) 17.60 ± 7.67 (n = 8) 15.56 ± 7.45 (n = 8) 15.70 ± 2.83 (n = 8)

Total content of fatty acids represented as the percent of all fatty acids, mean ± SD

C16:0 (palmitic acid) 19.649 ± 0.549 15.025 ± 5.828 17.250 ± 1.450 18.027 ± 1.070

C18:0 (stearic acid) 18.579 ± 1.388 20.020 ± 2.261 19.708 ± 1.597 18.172 ± 1.733

C18:1ω-9 (oleic acid) 8.760 ± 2.396 9.051 ± 4.611 11.055 ± 3.370 12.135 ± 2.383

C18:2ω-6 (linoleic acid) 28.425 ± 1.399 24.762 ± 3.306 21.430 ± 1.667 20.238 ± 1.758

C18:3ω-3 (α linolenic acid) 0.485 ± 0.261 0.209 ± 0.045 0.202 ± 0.214 0.186 ± 0.117

C20:4ω-6 (arachidonic acid) 8.290 ± 0.996 13.549 ± 1.924 10.490 ± 2.148 13.582 ± 2.899

C20:5ω-3 (eicosapentaenoic acid) 1.568 ± 0.122 0.616 ± 0.290 2.817 ± 0.594 0.582 ± 0.182

C22:5ω-3 (docosapentaenoic acid) 0.640 ± 0.087 0.391 ± 0.083 0.951 ± 0.108 0.360 ± 0.049

C22:6ω-3 (docosahexaenoate) 5.186 ± 0.687 5.945 ± 0.947 6.680 ± 0.717 6.562 ± 0.464

Fatty Acid Sham vs Sham SBFE 30% vs Sham SMOF 30% vs Sham OOFE 30% vs Sham

Ratio of fatty acids compared with value in sham or enterally fed group

C16:0 1 0.76 0.88 0.92

C18:0 1 1.08 1.06 0.98

C18:1ω-9 1 1.03 1.26 1.39

C18:2ω-6 1 0.87 0.75 0.71

C18:3ω-3 1 0.43 0.42 0.38

C20:4ω-6 1 1.63 1.27 1.64

C20:5ω-3 1 0.39 1.8 0.37

C22:5ω-3 1 0.61 1.49 0.56

C22:6ω-3 1 1.15 1.29 1.27

OOFE, 80% olive oil–supplemented fat emulsion; SBFE, standard soybean-based fat emulsion; SMOF, combination of soybean oil, medium-chain

triglycerides, olive oil, and fish oil emulsion.

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Feng et al 11

consist of. Although it is possible that a mouse may be less

tolerant of this higher lipid dose, we felt that the greatest influ-

ence on survival and the proinflammatory state was due to the

type of lipid, rather than the absolute dose. Whether the present

findings will translate to a clinical setting remains to be deter-

mined. As an example, a recent double-blind, randomized,

clinical trial comparing soybean oil vs olive oil FE in an adult

intensive care setting failed to demonstrate any differences in

survival or a reduction in infectious complications.35 In con-

trast, a recent systematic review suggested that fish oil lipid

emulsions resulted in improved infectious outcomes in surgical

patients.36 Use of a combination FE, SMOF, led to similar if

not superior results in a prospective controlled double-blind

study.37 Presently, only 1 class of IVFEs is available in the

United States, with all being soybean-based products, whereas

the other 2 FEs studied in this article are available in other

regions of the world, including Europe, Asia, the Middle East,

and South America. Based on the findings of this study and

many other lines of animal and clinical studies, it is increas-

ingly evident that newer forms of FE need to be studied and

used in the United States. Such a position recently has been

taken by the American Society for Parenteral and Enteral

Nutrition, and it is hoped that such efforts will lead to improved

nutrition care for those patients receiving PN.38 One limitation

of this study was that we confined our expression of cytokine

abundance and growth factors to the mRNA level. To take this

to the protein level would far exceed the scope of the present

study, but it will be important to carry this further in future

investigations. Another limitation was that we focused on 30%

of nutrients being delivered as an FE. Although this may be

clinically relevant to humans, it is higher than what most

rodents use on a daily basis. Thus, a perfect correlation with

the use of this model is not completely possible.

The implications of this work are considerable in that the

clinical application and selection of one type of IV lipid over

another is not readily clear, and the insights provided here

might help a clinician select one form over another or decide in

which clinical setting a particular FE may be most appropriate.

Clearly, these results are confined to the intestine, and a broader

examination of the physiologic effects of these lipids will need

to be performed.

References

1. Hippalgaonkar K, Majumdar S, Kansara V. Injectable lipid emulsions-

advancements, opportunities and challenges. AAPS PharmSciTech.

2010;11(4):1526-1540.

2. Iba T, Yagi Y, Kidokoro A, Ohno Y, Kaneshiro Y, Akiyama T. Total

parenteral nutrition supplemented with medium-chain triacylglycer-

ols prevents atrophy of the intestinal mucosa in septic rats. Nutrition.

1998;14(9):667-671.

3. Ulrich H, Pastores SM, Katz DP, Kvetan V. Parenteral use of medium-

chain triglycerides: a reappraisal. Nutrition. 1996;12(4):231-238.

4. Gogos CA, Zoumbos N, Makri M, Kalfarentzos F. Medium- and long-

chain triglycerides have different effects on the synthesis of tumor necro-

sis factor by human mononuclear cells in patients under total parenteral

nutrition. J Am Coll Nutr. 1994;13(1):40-44.

5. Waitzberg DL, Torrinhas RS. Fish oil lipid emulsions and immune

response: what clinicians need to know. Nutr Clin Pract. 2009;24(4):

487-499.

6. Carpentier YA, Dupont IE. Advances in intravenous lipid emulsions.

World J Surg. 2000;24(12):1493-1497.

7. Waitzberg DL, Torrinhas RS, Jacintho TM. New parenteral lipid emul-

sions for clinical use. JPEN J Parenter Enteral Nutr. 2006;30(4):351-367.

8. Meisel JA, Le HD, de Meijer VE, et al. Comparison of 5 intravenous

lipid emulsions and their effects on hepatic steatosis in a murine model. J

Pediatr Surg. 2011;46(4):666-673.

9. de Meijer VE, Gura KM, Le HD, Meisel JA, Puder M. Fish oil–based

lipid emulsions prevent and reverse parenteral nutrition-associated

liver disease: the Boston experience. JPEN J Parenter Enteral Nutr.

2009;33(5):541-547.

10. Feng Y, McDunn JE, Teitelbaum DH. Decreased phospho-Akt signaling

in a mouse model of total parenteral nutrition: a potential mechanism for

the development of intestinal mucosal atrophy. Am J Physiol Gastrointest

Liver Physiol. 2010;298(6):G833-G841.

11. Yang H, Finaly R, Teitelbaum DH. Alteration in epithelial permeability

and ion transport in a mouse model of total parenteral nutrition. Crit Care

Med. 2003;31(4):1118-1125.

12. Yang H, Sun X, Haxhija EQ, Teitelbaum DH. Intestinal epithelial cell-

derived interleukin-7: a mechanism for the alteration of intraepithelial

lymphocytes in a mouse model of total parenteral nutrition. Am J Physiol

Gastrointest Liver Physiol. 2007;292(1):G84-G91.

13. Wildhaber BE, Yang H, Tazuke Y, Teitelbaum DH. Gene alteration of

intestinal intraepithelial lymphocytes with administration of total paren-

teral nutrition. J Pediatr Surg. 2003;38(6):840-843.

14. Kiristioglu I, Teitelbaum DH. Alteration of the intestinal intraepithelial

lymphocytes during total parenteral nutrition. J Surg Res. 1998;79(2):

91-96.

15. Sun X, Spencer AU, Yang H, Haxhija EQ, Teitelbaum DH. Impact of

caloric intake on parenteral nutrition-associated intestinal morphology

and mucosal barrier function. JPEN J Parenter Enteral Nutr. 2006;30(6):

474-479.

16. Nose K, Yang H, Sun X, et al. Glutamine prevents total parenteral

nutrition-associated changes to intraepithelial lymphocyte phenotype and

function: a potential mechanism for the preservation of epithelial barrier

function. J Interferon Cytokine Res. 2010;30(2):67-80.

17. Feng Y, McDunn J, Teitelbaum D. Decreased phospho-Akt signaling in

a mouse model of total parenteral nutrition (TPN): a potential mechanism

for the development of intestinal mucosal atrophy. Gastroenterology.

2009;136(5)(suppl 1):A-96.

18. Yang H, Fan Y, Teitelbaum DH. Intraepithelial lymphocyte-derived

interferon-gamma evokes enterocyte apoptosis with parenteral nutrition in

mice. Am J Physiol Gastrointest Liver Physiol. 2003;284(4):G629-G637.

19. Feng Y, Sun X, Yang H, Teitelbaum DH. Dissociation of E-cadherin and

beta-catenin in a mouse model of total parenteral nutrition: a mechanism

for the loss of epithelial cell proliferation and villus atrophy. J Physiol.

2009;587(pt 3):641-654.

20. Xu Z, Harvey K, Pavlina T, Dutot G, Zaloga G, Siddiqui R. An improved

method for determining medium- and long-chain FAMEs using gas chro-

matography. Lipids. 2010;45(2):199-208.

21. Sun X, Yang H, Nose K, et al. Decline in intestinal mucosal IL-10

expression and decreased intestinal barrier function in a mouse model

of total parenteral nutrition. Am J Physiol Gastrointest Liver Physiol.

2008;294(1):G139-G147.

22. Feng Y, Teitelbaum DH. Epidermal growth factor/TNF-alpha transac-

tivation modulates epithelial cell proliferation and apoptosis in a mouse

model of parenteral nutrition. Am J Physiol Gastrointest Liver Physiol.

2012;302(2):G236-G249.

23. Yang H, Wildhaber B, Tazuke Y, Teitelbaum DH. 2002 Harry M. Vars

Research Award. Keratinocyte growth factor stimulates the recovery of

epithelial structure and function in a mouse model of total parenteral nutri-

tion. JPEN J Parenter Enteral Nutr. 2002;26(6):333-341.

at UNIV OF MICHIGAN on June 18, 2013pen.sagepub.comDownloaded from

12 Journal of Parenteral and Enteral Nutrition XX(X)

24. Yang H, Wildhaber BE, Teitelbaum DH. 2003 Harry M. Vars Research

Award. Keratinocyte growth factor improves epithelial function

after massive small bowel resection. JPEN J Parenter Enteral Nutr.

2003;27(3):198-207.

25. Sun X, Yang H, Nose K, et al. Decline in intestinal mucosal IL-10

expression and decreased intestinal barrier function in a mouse model

of total parenteral nutrition. Am J Physiol Gastrointest Liver Physiol.

2008;294(1):G139-G147.

26. Lanza-Jacoby S, Flynn JT, Miller S. Parenteral supplementation with a

fish-oil emulsion prolongs survival and improves rat lymphocyte function

during sepsis. Nutrition. 2001;17(2):112-116.

27. Lin MT, Hsu CS, Yeh SL, et al. Effects of omega-3 fatty acids on leuko-

cyte Th1/Th2 cytokine and integrin expression in rats with gut-derived

sepsis. Nutrition. 2007;23(2):179-186.

28. Gawecka A, Michalkiewicz J, Kornacka MK, Luckiewicz B, Kubiszewska

I. Immunologic properties differ in preterm infants fed olive oil vs

soy-based lipid emulsions during parenteral nutrition. JPEN J Parenter

Enteral Nutr. 2008;32(4):448-453.

29. Granato D, Blum S, Rossle C, Le Boucher J, Malnoe A, Dutot G.

Effects of parenteral lipid emulsions with different fatty acid composi-

tion on immune cell functions in vitro. JPEN J Parenter Enteral Nutr.

2000;24(2):113-118.

30. Feng Y, Ralls MW, Xiao W, Miyasaka E, Herman RS, Teitelbaum DH.

Loss of enteral nutrition in a mouse model results in intestinal epithelial

barrier dysfunction. Ann N Y Acad Sci. 2012;1258:71-77.

31. Yamaoka T, Yan F, Cao H, et al. Transactivation of EGF receptor and

ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis.

Proc Natl Acad Sci U S A. 2008;105(33):11772-11777.

32. Jacobi SK, Moeser AJ, Corl BA, Harrell RJ, Blikslager AT, Odle J.

Dietary long-chain PUFA enhance acute repair of ischemia-injured intes-

tine of suckling pigs. J Nutr. 2012;142(7):1266-1271.

33. Hayashi M, Tomita M. Mechanistic analysis for drug permeation

through intestinal membrane. Drug Metab Pharmacokinet. 2007;22(2):

67-77.

34. Li Q, Zhang Q, Wang M, Zhao S, Xu G, Li J. n-3 polyunsaturated fatty

acids prevent disruption of epithelial barrier function induced by proin-

flammatory cytokines. Mol Immunol. 2008;45(5):1356-1365.

35. Umpierrez GE, Spiegelman R, Zhao V, et al. A double-blind, randomized

clinical trial comparing soybean oil–based versus olive oil–based lipid

emulsions in adult medical-surgical intensive care unit patients requiring

parenteral nutrition. Crit Care Med. 2012;40(6):1792-1798.

36. Wei C, Hua J, Bin C, Klassen K. Impact of lipid emulsion containing fish

oil on outcomes of surgical patients: systematic review of randomized

controlled trials from Europe and Asia. Nutrition. 2010;26(5):474-481.

37. Klek S, Chambrier C, Singer P, et al. Four-week parenteral nutrition using

a third generation lipid emulsion (SMOFlipid)—a double-blind, ran-

domised, multicentre study in adults. Clin Nutr. 2013;32(2):224-231.

38. Vanek VW, Seidner DL, Allen P, et al. A.S.P.E.N. position paper:

clinical role for alternative intravenous fat emulsions. Nutr Clin Pract.

2012;27(2):150-192.

at UNIV OF MICHIGAN on June 18, 2013pen.sagepub.comDownloaded from